Circadian Activity And Expression Pattern Of Circadian Genes Of The Tree Shrew

Quantities of biological processes in mammals exhibit rhythms with approximate 24-hour periods,which were regulated by the core biological clock,as well as downstream clock controlled genes.However,most of previous studies about circadian rhythm were conducted in laboratorial nocturnal rodents,of which the results could not be completely applied on humans.As a representative of the order Scandentia,the tree shrew is evolutionarily related to the primates.Meanwhile,in the recent years the tree shrew has been applied in the research of human disorders for its human-like day-active characters,relative short gestation period and low feeding cost.However,few researches about the circadian system of the tree shrew have been reported,and the expression pattern of clock genes and downstream clock controlled genes in this species still remains unclear.In our present research,we focus on the circadian activity and expression pattern of circadian genes in the tree shrew.The main results are as follows:(1)Once more,we confirmed the day-active character of the tree shrew by recording their locomotor under normal light-dark circles.We found that the relative daytime activity of domestic female tree shrews was significantly higher than that of the males(p=0.040),and the activity onset of females was also significantly earlier than that of the males(p=0.002).We also provided a more detailed description of the morphology of BMAL1-labelled tree-shrew suprachiasmatic nucleus(SCN)via immunohistochemical staining and found that the volume of tree-shrew SCN was 3-4 times of the mouse SCN.Additionally,we determined the expression level of 12 core clock genes in different brain areas and peripheral tissues of the tree shrew via qPCR.The results indicated that the peripheral clocks,especially ARNTL and NPAS2,were much more robust as compared to the brain clocks.Meanwhile,CLOCK and CRY2 always exhibited weak rhythmicity in both brain and peripheral tissues.Additionally,there was a significant phase advance(2-4 h)for PER3,DBP and NR1D1 in the peripheral tissues as compared to the brain tissues,and the phase of CRY1 in female tree shrews was also phased-advanced by 3-4 h as compared to that of males.Furthermore,these clock genes exhibited an anti-phasic relationship between the diurnal tree shrew and the nocturnal mouse(i.e.,12-h phasing differential).(2)We obtained the transcriptome data of tree-shrew hypothalamus and cerebellum via high-throughput sequencing.Among the total 14721 detected genes,140 genes in male hypothalamus,539 genes in female hypothalamus,1280 genes in male cerebellum and 730 genes in female cerebellum exhibited significant oscillation.However,only 19 genes were shared among the four groups.We found lots of biological processes were rhythmic by using the KEGG pathway enrichment analysis,among which the protein processing in endoplasmic reticulum pathway was significantly enriched in the day and the ribosome biogenesis pathway was significantly enriched in the night.Gender difference also existed in the gene expression level of male and female transcriptomes.Most of the sex-biased genes,especially in hypothalamus,had high expression level in female tree shrews.Additionally,some hormone receptors(e.g.,adrenocortical hormone receptors and sex hormone receptor)and neuropeptides(e.g.,AVP and VIP)were inversely correlated with several circadian-regulatory genes in both male and female hypothalamus,which indicated potential sex difference in the regulation of circadian rhythm.Furthermore,we compared the tree-shrew circadian transcriptome with that of the nocturnal mouse and the diurnal baboon.The expression phase of circadian genes in the tree shrew was quite similar with that of the baboon and approximately anti-phasic with that of the mouse.Moreover,additional analysis of different transcriptome data from WT and BMAL1 KO mouse further supported the rhythmicity of the protein processing in endoplasmic reticulum pathway and the potential coordination of core clock genes with this pathway.(3)In the long photoperiod conditions,the locomotor activity of tree shrews showed two different patterns,namely delayed activity onset(DAO)and advanced activity offset(AAO).In the constant light conditions,the period of tree-shrew locomotor activity was shortened and a phase-advance was detected,accompanied by the phase-advance of core clock genes.However,the period of mouse locomotor activity was extended under constant light conditions,which resulted in a phase-delay of circadian activity and accompanied phase-delay of core clock genes.Moreover,constant light could enhance the rhythmicity of circadian genes to maintain the endogenous rhythms of the mice.In conclusion,the results of present study indicated that the conservation of circadian genes,and the expression pattern of circadian genes in the tree shrews was quite close to that of the diurnal primate,but also unique from the diurnal primate and nocturnal rodent.These findings may provide a molecular biological foundation for the research of diurnal rhythms,and promote the tree shrew to be the next potential animal model of diurnal rhythms.